Vaccination protocols have improved over the last several decades; however a therapeutically effective immune response has still been difficult to generate for some conditions. For example, human tumor immunotherapy has met with only limited success. Among the reasons for this difficulty have been the limited availability of tumor associated antigens, and an inability to deliver antigens in a manner that renders them immunogenic.
Dendritic cells (DC) include a heterogeneous family of antigen presenting cells (APC) that present antigens for the modulation of an immune response or induce immunological tolerance. The number of dendritic cells in the blood is surprisingly few, less than about 1% of blood mononuclear leukocytes. Thus, the low number of circulating dendritic cells makes their therapeutic use for the stimulation or modulation of an immune response difficult. Dendritic precursor cells, such as monocytes, migrate from a host's bone marrow to specific sites where they become activated and differentiate into dendritic cells. Following exposure to an antigen and an activation signal, the dendritic cells secrete chemokines and cytokines, and then present the processed antigen to naive T cells to produce an immune response in the host.
Bidirectional interactions between antigen presenting dendritic cells and naïve T cells initiate either an immunogenic or a tolerance pathway that are of particular importance in autoimmune disease and in transplantation medicine. Conventional subsets of dendritic cells described in humans include myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC).
Dendritic cells possess a distinct morphology and are characterized by the expression of large amounts of class II MHC antigens, and the absence of lineage markers, including CD14 (monocyte), CD3 (T cell), CD19, CD20, CD24 (B cells), CD56 (natural killer), and CD66b (granulocyte) (Shortman and Liu, Nat. Rev. Immunol. 2:151-161, 2002). Dendritic cells also express a variety of adhesion and co-stimulatory molecules such as CD80 and CD86, and molecules that regulate co-stimulation, such as CD40. The phenotype of dendritic cells varies with the stage of dendritic cell maturation and activation (Chapuis et al., Eur. J. Immunol. 27:431-441, 1997). However, expression of adhesion molecules, MHC antigens and co-stimulatory molecules increases with dendritic cell maturation. Antibodies that preferentially stain dendritic cells are commercially available, such as anti-CD83 and anti-CD80. Accordingly, the expression level of a particular antigen marker can be used to confirm if the antigen presenting cell is a dendritic cell, and if the dendritic cell is mature (Zhou and Tedder, J. Immunol. 154:3821-3835, 1995; Weissman et al., J. Immunol. 155:4111-4117, 1995).
Several in vitro methods have been developed to expand populations of dendritic cells and to augment anti-cancer immunity. Ex vivo exposure of expanded populations of dendritic cells to antigens found on tumor cells or other disease-causing cells, followed by reintroduction of the antigen-loaded dendritic cells to the subject, significantly enhanced presentation of the antigen to responding T cells. For example, culturing blood mononuclear leukocytes for eight days in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) was found to produce large numbers of dendritic cells (Sallusto and Lanzavecchia, J. Exp. Med. 179:1109-1118, 1994).
DNA vaccines that incorporate plasmids encoding cytokines (such as GM-CSF and IL-4) have been used to investigate dendritic cell maturation pathways. In particular, GM-CSF cDNA has been used as a DNA vaccine adjuvant for glycoprotein B of Pseudorabies virus (PrV) in a murine mouse model (Yoon et al, Microbiol. Immunol. 50:83-92, 2006). At least nine cytokine-secreting vectors have been identified as genetic adjuvants for DNA vaccines (in “DNA Vaccines Methods and Protocols,” edited by Douglas Lowrie and Robert Whalen).
Nucleic acid immunization is a relatively recent approach in vaccine development. The ability of DNA vaccines to protect against challenges from pathogens has been demonstrated in animal models of influenza, malaria, mycobacterium, HIV, and Ebola. A DNA-based vaccine usually comprises purified plasmid DNA carrying sequences encoding a target antigen under the control of a eukaryotic promoter. The plasmid is injected into the muscle or skin and the host cells take up the plasmid and express the antigen intracellularly. Expression of the encoded antigen by the host's cells is one of the advantages of this approach because it mimics natural infection. To enhance immune responses induced by DNA vaccines, co-administration of adjuvants such as cytokines, chemokines and co-stimulatory molecules have been used. It is therefore believed that administering plasmids encoding cytokines (such as GM-CSF or IL-4) and a target antigen may cause intracellular expression of both the antigen and the cytokine in the host, thereby providing an enhanced immune response in the host.
Cancers are a significant public health problem. Many cancer treatments are available to such patients, including surgical excision, chemotherapy, radiotherapy, and bone marrow transplantation. While many conventional cancer therapies are often effective in reducing neoplastic growth, healthy cells are frequently compromised by cytotoxic treatments. Non-selective cell damage causes pain, inflammation, hair loss, immunosuppression and gastrointestinal damage. Improved compositions and methods are needed to treat, inhibit, or alleviate the development of tumors. For example, tumor antigens have been administered to a tumor bearing host in attempts to produce an immune response to the tumor cells in the host. This approach has met with varying and modest results.
Granulysin is a naturally occurring protein expressed in human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Granulysin expressed in its full-length form has a molecular weight of approximately 15,000 Daltons and is known as 15 kD or 15 kDa granulysin. A post-translational modified form of 15 kD granulysin in which both the N- and C-termini are cleaved is known as 9 kD granulysin. The 9 kD granulysin peptide has been extensively studied and is observed to possess anti-microbial and tumorcidal activity (Hanson et al., Mol. Immunol. 36:413-422, 1999; Krensky, Biocehm. Pharmacol. 59:317-320, 2000; Clayberger et al., Curr. Opin. Immunol. 15:560-565, 2003; Deng et al., J. Immunol. 174:5243-5248, 2005; Stenger et al., Science 282: 121-125, 1998; and Huang et al., J. Immunol. 178:77-84, 2007). The 9 kD granulysin peptide is also known to have cytolytic properties and its resulting toxicity may limit its therapeutic use.